Clinical experience with adjunctive hyperbaric oxygen therapy in the treatment of diabetic ulcers has shown that wound hyperoxia increases wound granulation tissue formation and accelerates wound contraction and secondary closure. In addition to wound hyperoxia, increased wound nitric oxide production caused by hyperbaric oxygen therapy also appears to be important for successful diabetic wound repair.
The results of a preliminary retrospective study suggest that nitric oxide production is reduced in the nonhealing diabetic wound, and that topical becaplermin therapy is effective only when wound nitric oxide production deficiency is corrected. In addition, the data suggest that below a critical level of endogenous nitric oxide production, diabetic ulcer repair may not be achieved.
Under this hypothesis, diabetic patients with chronic, nonhealing ulcers that respond to becaplermin should have substantially increased endogenous nitric oxide production compared with those ulcers that do not respond to becaplermin.
The results of a preliminary clinical study support the use of combined therapy using topical becaplermin and hyperbaric oxygen therapy as a means of successfully treating the chronic diabetic ulcer patient with deficient nitric oxide production and local wound hypoxia.
ADV SKIN WOUND CARE 2000;13:769-74.
Although clinical and experimental studies continue to support the adjunctive use of hyperbaric oxygen (HBO) therapy to enhance wound healing?1-5 there is concern that present physiologic theories supporting HBO-mediated wound repair lack documentation. It is unclear whether the claims made for HBO-mediated wound healing improvements may be independently based on oxygen and pressurerelated phenomena. Proper oxygen availability for tissue metabolism is a crucial factor for collagen deposition and crosslinking,6 neutrophil-dependent microbial killing,7 and neovascularization.8
However, oxygen does not appear to play a primary role in the promotion of HBO-mediated phenomena, such as the inhibition of integrin-mediated intravascular leucocyte adhesions and platelet aggregation; enhancement of cutaneous microvascular homeostasis, as in recovery following ischemia-reperfusion syndrome; or the increased oxygen capacitance and survival of ischemic flaps.9 In these cases, endothelial modulation, cell-mediated inflammation, and wound matrix development have been enhanced or modified by the initiation of local regulatory mechanisms. If HBO-mediated wound hyperoxia were the single factor responsible for these processes, it would suggest that oxygen was capable of functioning both as a tissue metabolite and a cellular signal for wound repair.
Nitric oxide (NO) is a unique, gaseous free radical that is an important physiologic mediator for autonomic functions such as vasodilation, neurotransmission, and intestinal peristalsis. Recent wound healing studies of NO-mediated enhanced tissue repair10,11 and HBOmediated NO productions12-14 suggest physiologic mechanisms that may establish the dual role of oxygen during wound repair. Oxygen and L-arginine are combined by the enzyme nitric oxide synthase (NOS) to form NO and citrulline. Endothelial, cutaneous, and cellular sources of NO are important for successful wound repair.15 Therefore, NO-mediated cellular signaling may represent an additional oxygen-mediated, physiologic connection between HBO therapy and enhanced wound healing.
For the diabetic patient, an endogenous deficiency in the NOS enzyme leads to decreased wound NO production and a spectrum of related pathologies,16,17 such as impaired cutaneous vasodilation, decreased neurogenic vascular response, diabetic neuropathy, and endothelial cell dysfunction that inhibit the processes necessary for granulation tissue formation. These pathologies promote chronic diabetic ulcer (DU) development. Although the recombinant plateletderived growth factor becaplermin (rhPDGF-BB; REGRANEX Gel; OrthoMcNeil Pharmaceutical, Inc, Raritan, NJ) has been clinically successful for the treatment of DUs, it may not provide improved healing for patients with significantly decreased wound NO production.18 Because HBO therapy and becaplermin treatment may be associated with diabetic wound NO production, significant improvement in the clinical approach of managing DUs may be realized when combining these therapies.
Mechanisms of HBO Therapy
Recent reviews of HBO therapy for DUs have documented a significant enhancement of wound fibroblast activity and granulation tissue formation not seen with other treatments.19-24 These studies and others demonstrate increased granulation tissue formation as a function of increased fibroblast stimulation in the diabetic wound and increased collagen deposition caused by HBO treatment.25 Successful wound closure has been achieved by secondary intention and contraction or by use of an autograft of the wound following HBO treatments. Because of this, the selective clinical application of adjunctive HBO therapy for DU management has become a reliable and cost-saving strategy in the majority of cases.26-30
Hyperbaric oxygenation is achieved when a patient breathes 100% oxygen in an environment of elevated atmospheric pressure. Physiologically this produces a directly proportional increase in the plasma volume fraction of transported oxygen that is readily available for cellular metabolism. Arterial PO^sub 2^ elevations to 1500 mm Hg or greater are achieved with 2 to 2.5 absolute atmospheres of pressure (ATA), with soft tissue and muscle PO^sub 2^ levels elevated to about 300 mm Hg. This significant level of hyperoxygenation allows for reversal of the localized tissue hypoxia that may be secondary to ischemia or other local factors in the compromised wound.1,2,9
A key factor in HBO's enhancement of the hypoxic wound environment is its ability to provide adequate oxygen availability within the vascularized connective tissue compartment that surrounds a wound.9 Normally the hypoxic wound space is separated from the surrounding vascularized connective tissue compartment by a significant oxygen gradient. Proper oxygenation of the vascularized connective tissue compartment becomes an important rate-limiting factor for cellular functions associated with successful wound healing. The neutrophil, fibroblast, macrophage, and osteoclast have specific inflammatory and repair functions that are dependent on an environment that is not oxygen deficient. For each cell type, a wound PO^sub 2^ less than 40 mm Hg is associated with decreased or deficient cellular activity.9,31 Experimental studies of wound oxygenation also confirm that tissue PO^sub 2^ levels greater than 40 mm Hg are required for normal tissue repair to proceed.9,31 Current HBO patient selection algorithms continue to use this criteria in wound assessment.
Sequential wound hyperoxygenation by HBO gradually promotes wound angiogenesis,14,19,22,24,32 granulation tissue formation,14,19,22,24,31 and wound contraction.33 For these reasons, wound hyperoxia is an important primary mechanism for enhanced repair in DUs that demonstrate progressive healing with HBO therapy.
The role of NO as an important cellular signal for wound repair and endothelial-mediated vasomotor responses has been well established. However, the relationship between HBO-mediated increased cellular NO production and enhanced tissue repair has only recently been examined. Thom13 was able to correlate increasing treatment pressures of HBO therapy to a progressive, temporary inhibition of adhesion of activated neutrophils to the endothelial surface of the cerebral microcirculation in experimental models of carbon monoxide poisoning. In another study HBO-mediated NO impairment of polymorphonuclear leukocyte membrane B2-integrin receptors was shown to be responsible for the prevention of leukocyte-endothelial adhesions.34 Studies of alterations in central nervous system (CNS) oxygen metabolism by HBO have demonstrated an increased cellular production of oxidative radicals.12,35,36 Although not associated with tissue repair processes, increased oxygen-dependent norepinephrine metabolism and NO synthesis have also been suggested to be mutually active mediators of CNS oxygen toxicity following HBO therapy.12
A recent clinical study of chronic DU patients receiving HBO therapy showed a direct relationship between significantly increased wound epithelial cell accumulation of the NO metabolite 3nitrotyrosine (3-NI) and increased wound granulation tissue formation.14 Epithelial 3-NT accumulation of progressively healing ulcers was significantly elevated after the first 2 weeks of HBO treatment when compared with 3-NT levels in ulcers that showed minimal improvement. This suggests that: (1) increased wound NO production is an early event in wound repair, (2) effective clinical HBO therapy is associated with increased production of NO in the chronic wound, and (3) increased HBO-mediated wound NO production may enhance vasodilatation, decreased leucocyte and platelet adherence, and protection of the cutaneous microcirculation. It has been shown that increased NO activity at the perivascular interface also can exert feedback inhibition on NOS to control its synthesis from the endothelial cell.37 However, in the DUwhere NOS activity is deficient-physiologic maintenance of vasomotor tone, cell adhesion, and oxygen availability are compromised. In the chronic DU, HBOmediated increased NO production may promote microvascular recovery and normal wound repair. Although scientific verification of these processes is forthcoming, it appears likely that clinical HBO therapy is capable of significantly increasing wound NO production, thereby providing an additional primary mechanism for enhanced repair of the chronic hypoxic or diabetic wound.
The Role of Nitric Oxide in Wound Repair
Through cell-to-cell communications, NO activates its target molecule, guanylate cyclase, which elevates intracellular concentrations of cyclic guanosine monophosphate (cGMP).38 Increased cGMP causes vascular smooth muscle relaxation, which constitutes a significant mechanism of homeostasis for the microcirculation and modulates the cardiovascular response to vasoconstrictors, cytokines, and endotoxin. Nitric oxide may alter key enzymes, affecting subcellular systems, the Krebs cycle, or RNA/ DNA synthesis. This activity is performed without the need for cell surface receptor stimulation, or signal transduction. Nitric oxide crosses cell membranes without mediation of channels or receptors-it diffuses across cellular membranes isotropically. Because of its high diffusion coefficient, short half-life of about 5 seconds, and prompt decomposition, NO is ideal for its ability to act as a cellular signal for wound repair.
Nitric oxide is routinely generated in biologic tissues by 3 isoforms of NOS that metabolize L-arginine and molecular oxygen to citrulline and NO. Two of the 3 isoforms are constitutive enzyme systems (cNOS) that are described in neuronal cells (nNOS) and in endothelial cells (eNOS).39 With these isoforms, increased levels of intracellular calcium activate the enzymes via calmodulin. The calcium-dependent cNOS systems produce low, or picomolar, quantities of NO. The third system is the inducible isoform (iNOS), which is calcium independent. Expression of iNOS is controlled by tissue-specific stimuli, such as inflammatory cytokines, or by exogenous materials, such as bacterial lipopolysaccharide (LPS).
Once induced, production of NO within tissue can increase as much as 1000fold, forming an environment that is toxic to invading microorganisms. It appears that the cNOS enzymes are involved in maintaining skin homeostasis and providing regulatory function.39 The iNOS enzymes appear to be mainly associated with the inflammatory and immune responses that are also implicated in certain skin diseases such as psoriasis. In humans, keratinocytes, fibroblasts, and endothelial cells possess both the cNOS and iNOS isoforms. Wound macrophages and keratinocytes possess the iNOS isoform. In one wound healing study NO synthesis was shown to occur for 10 to 14 days after wounding and macrophages appeared to be the major cellular source.11
As a mediator of tissue repair, NO has been shown to promote angiogenesis40 and cellular migration41; increase wound collagen deposition and collagen crosslinking10; regulate vasodilatation15; inhibit platelet aggregation38; inhibit endothelial-leukocyte cell adhesions42; modulate endothelial proliferation and apoptosis43; increase the viability of random cutaneous flaps44; and enhance cellular immunomodulation and bacterial cytotoxicity.38 Because of these relationships, NO-mediated cellular signaling and cytotoxicity may provide enhancement to wound repair by increasing tissue oxygen availability and by promoting the inflammatory mediation of repair mechanisms and wound matrix development and remodeling. This influence of NO on the wound healing process appears to continue over the time required for tissue repair to be completed (Figure 1). In addition to the chronic wound, growth factors and tissueengineered skin substitutes also may be dependent upon NO mediation to promote successful tissue repair. Despite documentation of simultaneous cytotoxic and protective activities within the wound environment, NO provides substantial promotion of tissue repair and maintenance of the microcirculation.44
The major metabolic pathway for NO is to nitrate and nitrite, which are stable metabolites within tissue, plasma, and urine.38 In the presence of superoxide, NO production causes increased levels of peroxynitrite production and secondary protein nitration, leading to 3-NT accumulation as a metabolite-and biologic marker-of oxidative radical activity. Tracer studies in humans have demonstrated that perhaps 50% of total body nitrate and nitrite originate from the NO synthesis substrate of L-arginine.45,46 Although nitrate and nitrite are not measures of biologically active NO, plasma and urine samples can be obtained from individuals on a controlled low-nitrate and low-arginine diet after a period of fasting, allowing measurements of nitrate and nitrite values that can serve as an index of alterations in NO production.47
Nitric Oxide and Diabetic Wound Healing
The study of DUs represents a unique example of impaired wound healing characterized by an NO deficiency caused by a systemic deficiency in NOS activity. Clinical and experimental studies of DUs show a delayed inflammatory response, deficient granulation tissue, inhibited collagen synthesis, an impaired neurogenic response, and neuropathy that appear to be associated with wound NO deficiency. 10,11,14,17,22 Similarly, endogenous and wound NOS deficiency has been documented with steroid administration,48 and depressed cellular NOS expression has been observed in hypoxic endothelium.49
In addition to having decreased NO production, diabetic wounds have reduced expression of growth factor receptors during wound repair. Experimental studies suggest that a certain level of platelet-derived growth factor (PDGF) and its receptors are essential for normal tissue repair.50 Platelet-derived growth factor stimulates cellular chemotaxsis of inflammatory cells and repair cells to the site of injury. In addition, smooth muscle cell and endothelial cell proliferation are promoted by PDGF.51 Increased wound tensile strength, granulation tissue formation, epithelialization, and angiogenesis have occurred following becaplermin treatments.52 In one study, however, successful wound repair was reported in less than 50% of cases following the clinical application of becaplermin gel to diabetic neuropathic ulcers. This finding suggests that other factors may be involved.53
A Retrospective Study
In a retrospective study of DU patients using becaplermin, the author and colleagues measured the plasma and urinary NO metabolites nitrate and nitrite of 10 DU patients without cardiovascular, renal, or infectious complications, and 10 healthy controls.18 The subjects were observed in a hospital inpatient environment and given a low-nitrate, lowarginine diet for 24 hours. Fasting urine and serum samples were obtained at the beginning and end of this period. Half (n = 5) of the DU patients had experienced successful healing with becaplermin (healed), and half (n = 5) had failed to heal (unhealed), despite equivalent becaplermin treatment. Both groups consisted of diabetic neuropathic patients with a history of foot ulceration but no peripheral vascular disease. All ulcers were surgically debrided prior to becaplermin application.The groups were similarly matched for age, male-to-female ratio, type of diabetes, initial ulcer size, weight, body mass index, glycosylated hemoglobin level, serum creatinine, and creatinine clearance.
Data analysis showed significantly decreased (P
These findings suggest that DUs that do not respond to becaplermin experience significantly lower nitrate metabolism. This may reflect a substantially reduced level of endogenous NO activity. In addition, the data suggest that critical levels of endogenous NO activity are required for successful DU healing. Future clinical surveillance of NO, or its metabolites, may enable accurate predictions of patients that will have successful outcomes with becaplermin treatment.
The observations of the retrospective study imply a symbiotic relationship among wound NO production, PDGF activity, and successful DU healing that has not been previously reported. As clinicians attempt to institute a new paradigm for impaired wound healing of patients with diabetes, they should also reexamine current guidelines for HBO and becaplermin treatment. Clinical indications for HBO therapy for the chronic wound or DU should not be limited to the evidence of hypoxia alone, but should also consider the evidence of deficient endogenous or wound NO production as an indication for treatment. For the steroid-dependent ulcer patient, indications for HBO therapy may be similarly applied. This treatment may prove beneficial in the regeneration of healthy granulation tissue formation and the promotion of secondary wound closure in the absence of detectable wound hypoxia. For the DU patient, wound NO metabolite measurements may also serve as biologic monitors for patient assessment when topical becaplermin gel is considered. In addition, fasting serum and urine nitrate levels and wound NO metabolite assays may provide valuable information for ensuring effective HBO wound stimulation and for determining the end point of HBO treatment. Dietary supplementation of L-arginine may provide an alternative to HBO therapy, depending on the degree of NO deficiency and local wound pathology.54
The results of the retrospective clinical study support the use of combined therapy using topical becaplermin and HBO therapy as a means of successfully treating chronic DU patients with deficient NO production and local wound hypoxia. Continued investigations of endogenous and wound NO production as a predictive factor in the management of DUs are warranted. Current studies in this area may offer clinicians a novel physiologic approach to the understanding of impaired diabetic wound healing.
Additional clinical guidelines for DU patients considered for becaplermin treatment also should be developed. Similarly indications for HBO therapy for chronic steroid-dependent wounds or DUs should be expanded and modified to reflect abnormalities in endogenous or wound NO production. Additional clinical criteria would provide for better patient selection for this combined treatment, improved surgical preparation of the hypoxic or diabetic ulcer, and improved clinical outcomes. Although the technology to support this clinical strategy is available, additional work is needed to break the costly cycle of chronic ulceration, recurrent infection, tissue loss, and amputation that plagues the DU patient.
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Joseph V. Boykin, Jr, MD, is the Medical Director of Columbia Retreat Hospital Wound Healing Center, Richmond, VA. Submitted January 21, 2000; accepted March 13, 2000.
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